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Abstract

Background

The Limbal epithelial crypt (LEC) is a solid cord of cells, approximately 120 microns
long. It arises from the undersurface of interpalisade rete ridges of the limbal palisades
of Vogt and extends deeper into the limbal stroma parallel or perpendicular to the
palisade. There are up to 6 or 7 such LEC, variably distributed along the limbus in
each human eye.

Morphological and immunohistochemical studies on the limbal epithelial crypt (LEC)
have demonstrated the presence of limbal stem cells in this region. The purpose of
this microarray study was to characterise the transcriptional profile of the LEC and
compare with other ocular surface epithelial regions to support our hypothesis that
LEC preferentially harbours stem cells (SC).

Results

LEC was found to be enriched for SC related Gene Ontology (GO) terms including those
identified in quiescent adult SC, however similar to cornea, limbus had significant
GO terms related to proliferating SC, transient amplifying cells (TAC) and differentiated
cells (DC). LEC and limbus were metabolically dormant with low protein synthesis and
downregulated cell cycling. Cornea had upregulated genes for cell cycling and self
renewal such as FZD7, BTG1, CCNG, and STAT3 which were identified from other SC populations. Upregulated gene expression for growth
factors, cytokines, WNT, Notch, TGF-Beta pathways involved in cell proliferation and
differentiation were noted in cornea. LEC had highest number of expressed sequence
tags (ESTs), downregulated and unknown genes, compared to other regions. Genes expressed
in LEC such as CDH1, SERPINF1, LEF1, FRZB1, KRT19, SOD2, EGR1 are known to be involved in SC maintenance. Genes of interest, in LEC belonging to
the category of cell adhesion molecules, WNT and Notch signalling pathway were validated
with real-time PCR and immunofluorescence.

Conclusions

Our transcriptional profiling study identifies the LEC as a preferential site for
limbal SC with some characteristics suggesting that it could function as a 'SC niche'
supporting quiescent SC. It also strengthens the evidence for the presence of "transient
cells" in the corneal epithelium. These cells are immediate progeny of SC with self-renewal
capacity and could be responsible for maintaining epithelial turn over in normal healthy
conditions of the ocular surface (OS). The limbus has mixed population of differentiated
and undifferentiated cells.

Background

Corneal transparency is crucial for sight. The corneal epithelium and tear film provide
the polished outer surface to the cornea enabling it to function as a refractive surface.
It is postulated that the continued supply of the epithelial cells is maintained by
the SC at the limbus. Numbers of studies have provided direct and indirect evidence
to support this notion. In 1986 Schermer et al proposed the limbal location of corneal
stem cells based on keratin expression of corneal epithelial cells [1]. Other studies providing evidence of presence of corneal stem cells at limbus include
immunohistochemistry studies with known SC markers [2-4], cell cycling studies characterising the limbal-basal epithelium [5,6] and electron microscopic features of the basal epithelial cells [7]. We have identified a unique structure at the limbus, termed as the limbal epithelial
crypt (LEC) [8]. It is a solid cord of cells which extends from the peripheral aspect of the undersurface
of interpalisade rete ridges of limbal palisades of Vogt into the limbal stroma (Figure
1A). There are up to 6 or 7 such LEC, variably distributed along the limbus in each
human eye. The LEC is analogous to the deep ridges reported in the monkey palm epithelium,
where the basal cells of the deep ridges are shown to be the slow cycling stem cells.
Similar to the deep location of the ridges in the monkey palm, the deep location of
the LEC would offer physical protection to the SC population [9,10]. Our anatomical and immunohistological studies on the LEC have emphasised its potential
as a repository of SC and as a putative SC niche (SCN) [11,12], a concept first proposed by Schofield in 1978 [13]. Constituents of the niche include tissue cells, and extra-cellular substrates that
sustain the SC and control their self renewal and progenitor potential in vivo [14]. The niche provides a specialised microenvironment whereby SC are maintained in a
state of quiescence. Cellular quiescence indicates slow cell cycling or growth arrested
phase of the cells. In adult SC it protects against environmental stresses and aids
in their maintenance. This property was identified in various cell populations such
as in the bulge region of hair follicles [15], intestinal [16], haematopoietic [17], muscle satellite SC [18] and also in the limbus [19]. In the hair follicle bulge region two SC compartments have been identified [15]; the quiescent and the activated progenitor cells. The latter regenerates the tissue
in homeostatic conditions whereas; quiescent cells act as a reservoir and undergo
cell cycling following tissue injury. Several studies have identified possible SC
markers in the limbal epithelium, using a mechanical dissection technique [20-22]. This method potentially involves the risk of contamination from surrounding tissue.
However, laser microdissection (LMD) has allowed the isolation of a pure population
of both limbal and corneal epithelium in situ [23]. Studies have also been performed on limbal sub-populations using different techniques
including cell cultures [24], collagen adhesiveness [25] and flow cytometry [26]. However such methods involving cellular manipulation can influence gene expression.
This transcriptional profiling study of laser microdissected OS epithelial regions
(LEC, limbus, cornea, conjunctiva and LEC stroma) demonstrates the average characteristic
features of each region rather than of the individual cell populations. Broadly, this
study highlights the presence of undifferentiated and quiescent SC in the LEC and
"transient cells" or activated progenitor cells and differentiated cells in the cornea
[27]. The gene expression of limbus is suggestive of presence of quiescent cell population
with differentiated suprabasal epithelial cells. Our study provides evidence to support
the hypothesis that the LEC is the reservoir of the SC and could serve as a SC niche
at the human OS.

Figure 1.Laser Microdissection (LMD) of the ocular surface epithelial regions. The composite shows steps of LMD performed on radial cut section of LEC (A, B,),
limbus (C, D), cornea (E, F), conjunctiva (G, H) and LEC stroma (I, J) at 20× magnification
(scale bars shown). Figure 1A is of pre LMD LEC, shown with black arrow. Figure 1
I is of LEC stroma with cells shown with white arrow. Prior to LMD, the sections were
stained with RNase free Toluidine Blue. Images (A, C, E, I,) are examples of pre LMD
OS epithelial region sections with outlines for laser cuts drawn around the tissue.
The junctions between the OS epithelial regions were avoided as it has overlapping
features of the two adjacent regions. Image (G) shows cut sections of the epithelial
regions. Dividing the epithelium into multiple small pieces facilitated effective
catapulting of the tissue into the collection cap. Images (B, D, F, H, J) represent
examples of post LMD sections of OS epithelium following pressure catapulting of the
epithelial pieces. Image J shows a misdirected piece of LEC Stroma which was not captured
in the cap but settled down over the adjacent LEC Stroma (black arrow head); such
tissue pieces could be recatapulted into the cap with dot LPC laser function.

Results and Discussion

Transcriptome analysis of OS regions; 1) LEC, 2) limbus, 3) cornea, 4) conjunctiva
and 5) LEC stroma was performed on four biological replicates for each region, processed
from four pairs of cadaver human eyes. Poor quality raw data from a corneal and a
conjunctival replicate were excluded from the analysis. Following normalisation and
filtration, a data set of 4574 genes for 18 samples was created.

Differentially Expressed Gene Lists

Principal Component Analysis (PCA) grouped the biological replicates for each OS region.
Figure 2 (left) shows LEC replicates segregated from other OS regions. This demonstrated similarity
and reproducibility amongst the LEC biological replicates. Notably, PCA of differentially
expressed genes between LEC and cornea generated distinct gene clusters (figure 2, right) as these two regions are at the opposite ends of the epithelial differentiation
spectrum. The differentially expressed gene list for each OS region demonstrated highest
upregulated gene expression in the cornea and most downregulated genes in LEC. Our
findings showed that cornea was the most biologically active zone, whereas the LEC
was a metabolically dormant zone (table 1).

Figure 2.Principal Component Analysis (PCA) plot of microarray samples and genes. Left image shows PCA of LEC vs ALL samples following feature subset analysis performed on Jexpresspro software. The
LEC samples are represented as green dots and rest of the samples as blue dots. LEC
biological replicates are seen to cluster separately from rest of the samples indicating
differences between LEC and other sample groups but similarity or reproducibility
between LEC replicates. Right image shows PCA of differentially expressed genes (527)
between LEC and cornea. These are clustered into four distinct coloured groups according
to the density. The red group has the highest density and blue the lowest, black dots
in the centre are unclustered genes.

Gene Ontology (GO)

Considering the model of corneal epithelial regeneration [12,28-30], comparative GO profiling of LEC, limbus and cornea, was performed with the GO terms
categorised according to specific functions [31] related to stem, transient amplifying (TAC) and differentiated cells (DC) [32,33]. SC related GO terms enriched in LEC and limbus were Transcription from RNA polymerase II promoter, regulation of transcription from RNA polymerase II promoter and RNA binding. Compare analysis performed on Ingenuity pathway analysis (IPA), characterised the
'tissue specific' nature of each region. LEC was found to be enriched for undifferentiation
processes such as System development (4.2E-04), including spermatogenesis (SLC12A2), development of haematopoietic progenitor cells (EGR1) and nervous system development (LEF1, EGR1, PARK2, SLC12A2). This suggests that the LEC is a more undifferentiated
and stem-like region than the adjoining limbus. Also down regulation of genes related
to TAC terms, such as cell proliferation and apoptosis in LEC demonstrates its primitive features, unlike limbus. Meta-analysis performed
by Edwards et al on SC microarray data sets have identified GO terms related to quiescent
and proliferating SC [33]. Based on this study we have found that LEC was specifically enriched for quiescent
SC (QSC) related terms such as Biological Regulation, (p value: 4.0E-2), and Regulation of cellular processes (p value: 5.0E-2) (figure 3A). However limbus [Protein folding (p value: 2.6E-2)] and cornea [Primary metabolic process (p value: 7.1E-5), Translation (5.5E-11)] were enriched for PSC related GO terms. Additionally cornea and limbus
were enriched for TAC and DC related GO terms (figure 3B).

i) Transcription factors are involved in development and SC functions such as regulating cell fate determination,
cell cycling, cell differentiation and response to environment. This GO term was significant
only to LEC (p value: 3.50-05) and limbus (p value: 5.70E-04), (figure 3A, table 2). LEF1 has been shown to be crucial in hair follicle patterning [35] epithelial invagination into mesenchyme [36], and in maintenance of SC quiescence [37,38], it was found to be upregulated in LEC. In contrast, the upregulated transcription
factors in limbus were involved in cell proliferation and differentiation (table 2). A study on embryonic limbus has demonstrated expression of SOD2 (transcription factor and antioxidant) exclusively in limbus from 14 weeks of development
[39]. However our microarray study had noted upregulated expression of SOD2 in LEC, limbus
and cornea (table 3)

ii) Self renewal is an important feature of SC maintenance, which isabsent in differentiated cells,
weakly expressed in quiescent SC but significantly expressed in proliferating progenitor
cells such as mesenchymal stem cells (MSC), [40], intestinal crypts and neural crest cells[41,42]. Based on other SC studies such as adult SC [43], MSC [44], neural SC [45] and neural progenitor cells[46] we had found self renewal genes to be predominantly upregulated, in cornea (table
2). This is indicative of the presence of early TACs, (Transient cells), with self-renewal
properties in cornea. These cells could sustain healthy corneal epithelium independent
of limbal/LEC support. Dua et al [27] have demonstrated the long term conservation of 'central islands' of normal corneal
epithelium in patients with total limbal stem cell deficiency as determined clinically
by in vivo confocal microscopy. Study on cornea of certain mammals by Majo et al have demonstrated holoclone capacity of the central cornea and showed that the central
corneal epithelium, when transplanted to the limbus, could regenerate corneal epithelium
[47]. However Sun et al have refuted this and argued that true corneal epithelial SC are
located primarily at the limbus and not in the central cornea [48]. Data from this study supports the latter notion, and further refines the preferential
location of SC in the LEC. An ex-vivo study on organ cultured corneas has demonstrated
that the central corneal epithelium depicts a regenerative potential in acute wound
healing [49]. Some of these differences are clearly species dependent and any oligopotent potential
of human central cornea is not yet proven. Data from the above reports can be reconciled
by proposing that the SC are primarily located at the limbus/LEC and that the central
cornea contains cells that are capable of maintaining a sustained regenerative capacity
in the absence of a functional limbus, as well as in the uninjured physiological state.
This supports the hypothesis that the transition from a SC to a TAC is not abrupt
but that there exists a population of cells between these two stages that have an
intermediate potential, which we have termed "transient cells", and can migrate to
populate the central cornea[27]. In this study presence of enriched TAC related GO terms with significant gene expression
in cell cycling, self renewal, cell proliferation in cornea (table 2) is suggestive of the presence of such "transient cells" with self renewal capacity
in this region.

iii) Cell proliferation is a property of activated progenitor cells. Upregulated genes for cell proliferation
in cornea mainly belonged to TGFB1 and the Ras Oncogene family (table 2). Integrin Beta 1 Binding Protein 1 (ITGB1BP1) involved in disruption of focal cellular
adhesion and mobilisation of stem/transient cells via the c-Myc promoter [50] was found to be downregulated in LEC. This gene was further validated by real time
PCR. LYN a tyrosine kinase molecule involved in cell proliferation of haematopoietic stem cells
[51] was also found to be upregulated in limbus.

iv) Cell differentiation is a process whereby undifferentiated regenerative cells acquire specialised structural
and functional features of mature cells. It was enriched in all the 3 groups. Of the
sixty two, cell differentiation genes identified in LEC only five were upregulated
(table 2) these were involved in maintaining epithelial cells in an undifferentiated state.
However, in limbus of the 43 genes 23 were upregulated, out of which only 3 genes
were involved in maintaining the cells in undifferentiated state (KRT19, SOD2, KRT14) (table 2). KRT19 has been identified in epidermal stem/progenitor cell population with a role in negative
regulation of differentiation [52]. Lyngholm, M et al identified SOD2 as a marker of limbal SC [53], it was found to be upregulated in LEC, limbus and cornea in this study.

In cornea, of 113 genes expressed, 79 were upregulated and were involved in differentiation,
including terminal differentiation (table 2). Retinoic acid pathway involved in inhibition of proliferation of corneal TAC [54] was also expressed in cornea (RXRG, RARRES3, CYP26A1) (table 2). Epidermal Differentiation Complex (EDC) is a family of S100 related genes crucial
for terminal differentiation of human epidermis [55]. Of the 18 known S100 genes for EDCs four were upregulated in cornea and one in limbus
(table 2). Down-regulation of all these protein complexes was noted in LEC. Apolipoproteins
expressed in differentiated cells were found to be upregulated in cornea and absent
or downregulated in the LEC and limbus.

v) Negative regulation of cell proliferation, also known as 'cell quiescence', was enriched only in LEC (p value: 1.4E-2), and
LEC stroma (p value: 3.5E-2). SERPINF1 a secreted Neurotropin with potent antiangiogenic properties upregulated in early
passage cells in G0 phase as compared to actively proliferating or senescent cells
[56] was also found to be upregulated in LEC and limbus.

vii) Keratins: KRT19 a known epidermal SC marker [52] was also found to be upregulated in LEC and limbus; hence supporting the evidence
of presence of SC in these regions. However limbus also had upregulated expression
of KRT 14 which is expressed by mitotically active basal cells of stratified epithelium [57] and KRT 13 expressed by suprabasal cells of non-cornified stratified epithelia [58]

Molecular features of OS SC niche components

Adult SC are influenced by their microenvironment or the niche, which regulates their
function. Niche components identified in OS regions included cell adhesion molecules
(CAMs), growth factors, cytokines, extracellular matrix and secreted proteins like
neurotropins (table 3). E-cadherin, a CAM upregulated in LEC was previously shown to anchor the SC to the
basement membrane and thus aid in SC maintenance [59] and prevent aging of SC [60]. SIGLEC1 a CAM and a cell surface receptor, also upregulated in LEC, has been shown to be involved
in epithelial regeneration [61]. Growth factors and cytokines promote proliferation and these were found to be upregulated
in cornea (table 3). However IGFBP2 (Insulin Growth Factor Binding Protein 2) which is known to influence epidermal regeneration
and SC maintenance [62] was found to be upregulated in LEC. Protection of stem and progenitor cells from
oxidative stress is crucial for their sustained maintenance. The GO term detoxifier
system (GOTERM_MF_ALL: GO: 0016209) which confers antioxidant protection, was enriched in LEC (P value: 5.0E-2), cornea
(p value: 5.1E-3) and limbus (p value: 4.7E-2). SOD2 an antioxidant was found to be upregulated in LEC, limbus and cornea (table 3). It was previously shown to prevent the aging of the SC and their niches in human
epidermal keratinocytes [60]. We further validated the expression of SOD2 in these regions with real time PCR.

Unlike the cornea, the stroma of the limbus has been shown to maintain the basal epithelial
layer in an undifferentiated condition, preserving the stemness of the SC [63]. Previous studies have shown the presence of a heterogeneous population of cells
which include bone marrow derived mesenchymal cells in the limbal stromal region [64] along with the limbal epithelial progenitor cells that have migrated in the process
of epithelial mesenchymal transition (EMT) [65]. Similarly we had noted round undifferentiated epithelial and spindle like mesenchymal
cells in LEC stroma on histological sections of LEC (marked with white arrow head)
(Figure 1I). Therefore we further studied the gene expression profile encoding the GO term "secreted"
(extra cellular region GO: 0005576) for its influence on the LEC cells. The absence
of CDH1 in LEC stroma, along with activation of LEF1/WNT β-Catenin signalling pathways
in LEC could account for EMT which was similarly reported in a previous study on limbal
stroma [66]. LEC stroma was enriched for the tissue metalloproteinase inhibitors (TIMP1, TIMP2), developmental protein FLII (7.2), antioxidants XPA (4.57), DUOX1 (5.11), member of GDNF family GFRα4, and LMNA a nuclear envelope matrix protein, all of which have been reported to be involved
with SC maintenance in other tissues[67,68].

Unlike epithelium from different regions, we did not compare the LEC stroma with stroma
from other regions. This is a limitation of the current work; however, we were able
to compare the gene expression of LEC stroma with published data on limbal stroma
[63]. The above mentioned data on LEC stroma is supportive of its role in maintenance
of the LEC cells in undifferentiated state.

Canonical pathways on ocular surface

IPA was used to characterise the enriched canonical metabolic and signalling pathways
on the OS epithelial regions (table 4).

i). Metabolic pathways

Out of 41 molecules involved in energy metabolism in cornea, 35 were upregulated by
more than 2 fold indicating active metabolism in this region possibly related to cell
division and turn over. Likewise, Karsten et al [69] have noted upregulated expression of oxidative phosphorylation, purine and protein
metabolism in neural progenitor cells denoting increased energy consumption and high
protein turn over due to active cellular processes like proliferation and cell migration.
Cornea also had upregulated expression of related carbohydrate metabolic pathways
such as pyruvate, citrate/glycolysis and gluconeogenesis. Cornea was also enriched
for glutathione metabolism, which is crucial for maintaining corneal transparency,
cell membrane integrity and protection against oxidative stress.

Studies on side population cells from various tissues have demonstrated that the energy
consumption, transcription, translation and metabolism in the undifferentiated and
quiescent cells are minimal [24,70]. Although oxidative phosphorylation, amino acid, carbohydrate and energy metabolism
were found to be enriched in LEC, limbus and cornea, the gene expression in these
pathways was downregulated in LEC and limbus, which supports the presence of undifferentiated
and quiescent cells in these regions.

ii). Signalling pathways

All the three OS regions were enriched for NRF2 mediated oxidative stress response
(table 4). It has a role in cell protection during cell cycling.

SC signalling pathways in the OS

Fevr et al have demonstrated the importance of WNT receptor-beta catenin signalling pathway in
maintaining intestinal crypt structures and SC in their niche [71]. It is also crucial for maintaining HSC in a quiescent state and also has a role
in SC self renewal [72]. We had found upregulated expression of molecules, involved in WNT receptor-beta
catenin signalling such as LEF1, and CDH1 in LEC. Soluble WNT antagonists (sWA) maintain
skin bulge SC quiescence[73] and also contribute to SC pool maintenance in gastric tissue[41]. FRZB1, a soluble WNT antagonist (sWA) was uniquely expressed in LEC. FRZB1 was weakly
expressed in microarrays but real-time PCR and immunofluorescence results showed high
gene and protein expression of FRZB1 in LEC compared to other regions (Figure 4A, 5F). Planar polar component of WNT pathway, involved in regulation of cell adhesion
and motility, was upregulated in cornea only (table 5). Genes related to the Notch [74], Jak/STAT [75], TGF-Beta/BMP [76] and the Hedgehog (HH) signalling pathways involved in regulation of cell proliferation
and differentiation in response to cytokines and growth factors; were found to be
upregulated in cornea but not in LEC (table 5). HES1, a target gene of Notch signalling pathway, and RBX1 a TGF-Beta gene were
further validated with real time PCR.

Figure 4.Composite image of graphs of Real Time PCR performed on genes of interest. The real time PCR was performed on OS epithelial regions of LEC, cornea, limbus
and conjunctiva comprising of three biological replicates (3 eyes) with four sets
of technical replicates A, B, C & D. These were processed in triplicate. The cycle
threshold (Ct) value for these samples were averaged and normalised with 18S rRNA
Ct values. Significant p values between LEC and other OS regions is shown as (*),*p
< 0.05, ***p < 0.001. Data are expressed as means +/- standard errors of the mean
(SEM).

Comparison of gene expression in OS epithelium with other SC populations

LEF1 was found to be crucial for maintaining stemness across various SC populations
such as embryonic [77], mesenchymal [44] and epithelial [36] SC. In this study it was uniquely upregulated in LEC. Myc genes encode for transcription
factors, which activate genes influencing cell proliferation, cell growth, apoptosis
and SC self-renewal. A study on epidermal SC has noted upregulated expression of myc
genes in α6+/MHCI+ cells. These cells have characteristic features of TAC. However, myc genes were found
to be downregulated in α6+/MHCI- population of cells consisting of quiescent SC [78]. Myc genes such as ANXA1, TGFB1, FTH1, VAMP8, KRT5, HSPB1 and UGCG were upregulated in cornea and TXN (4.5) in limbus but these genes were downregulated
in LEC. Self renewal genes such as FZD7 [44,78-80], PCNA [69], and STAT3 [77,81-83] expressed in PSC populations (haematopoietic, mesenchymal, epithelial, neuronal and
embryonic SC) were also upregulated in cornea.

Comparison with other ocular gene expression studies

We had noted some similarities with other OS gene expression studies, particularly
with regards to KRT19, an epithelial SC marker previously identified in limbus [11]. KRT19 was found to be upregulated in LEC and limbal epithelium in this study. KRT13
a suprabasal epithelial marker [23], was also upregulated in limbus. A study comparing the gene profile of limbal and
corneal basal cells in mice has noted preferential expression of epithelial SC genes
such as FGFR1 (Fibroblast Growth Factor Receptor 1) and S100A6 (S100 calcium binding
protein A6) in limbus [23], however we had noted upregulation of these molecules in the corneal epithelium.
This difference could be related to an interspecies variation. We had also noted upregulated
expression of CRTAC1, CTSL2, NQO1, KRT12, MAL, IGFBP6, IGFBP7, S100A10 in the cornea. These genes were previously identified by Turner et al in their oligonucleotide
microarray study on corneal and conjunctival epithelium [84]

Validation of microarray data

i) Quantitative real-time PCR (qPCR)

Relative quantification of genes of interest (FRZB1, RBX1, INTGB1BP1, HES1, SOD2) was performed with real time PCR on OS regions such as limbus, cornea, and conjunctiva
in comparison with LEC (figure 4). FRZB1 was found to be significantly expressed in LEC but was absent in all corneal
replicates and insignificantly expressed in limbus and conjunctiva (figure 4A). RBX1 was significantly expressed in LEC compared to cornea and conjunctiva (figure
4B). Significant expression of ITGB1BP1 was noted between limbus and LEC with least
expression in LEC (figure 4C). Significant expression of HES1 was noted between LEC and cornea and also between
LEC and conjunctiva (Figure 4D). Although SOD2 was significantly expressed in all the OS regions posthoc analysis
had failed to demonstrate any significant relationship between the groups (Figure
4E).

ii) Immuno fluorescence

Immunofluorescence of frozen tissue sections of OS epithelium was performed with FRZB1 and HES1 antibodies (Figure 5). A previous study on HES1 expression in mice corneal epithelial stem/progenitor
cells has demonstrated that an increased expression of HES1 in these cells was crucial
for regulation of corneal development and homeostasis [85]. Intense nuclear staining of HES1 was noted in LEC and the stromal cells adjacent to LEC (figure 5A) as compared to limbus (figure 5B). This evidence is supportive of increased proportion of stem progenitor cells in
LEC and also in the surrounding LEC stroma. Few cells in the cornea along the basal
epithelium also expressed HES1 (Figure 5C). Nuclear staining with FRZB1 was prominently seen in the basal epithelium of the
LEC (Figure 5F) and in some areas of the limbus (Figure 5G). Figure 5H shows absence of FRZB1 expression in corneal epithelium.

In summary, gene ontology and gene expression patterns noted in this study are suggestive
of LEC being the most metabolically dormant and undifferentiated region as compared
to cornea and limbus. LEC was enriched for GO terms related to quiescent adult SC.
Upregulated genes in LEC such as CDH1, SERPINF1, LEF1, FRZB1, KRT19, SOD2, EGR1 along with Beta catenin-WNT signalling pathway are known to be involved in SC maintenance.
However limbus and cornea had presence of a mixed population of stem/progenitor, and
differentiated cells. Similar to LEC limbus was metabolically dormant, but also had
upregulated SC signalling molecules related to WNT, TGF-Beta and Hedgehog pathways.
Cornea had upregulated gene expression related to cell cycling, self renewal, proliferation,
cell differentiation growth factors, cytokines and SC signalling genes in the WNT,
Notch, TGF-Beta pathways. This further strengthens the evidence for the presence of
long term surviving early TACs (transient cells) with self renewal capacity in the
cornea.

Conclusion

This study is the first to characterise the in situ gene expression profile of laser microdissected LEC and demonstrate the presence of
two distinct SC compartments on the OS. We have demonstrated, at the transcriptome
level that the LEC has features that appear to be consistent with that of a quiescent
SC niche. Although the limbus was metabolically dormant it had a mixed population
of differentiated and undifferentiated cells. Our study clearly demonstrates that
cornea is the most differentiated and proliferating region. The gene expression of
this region is also suggestive of the presence of early TACs with self renewal capacity
(transient cells) Clinical evidence supports our findings that cornea has the potential
to sustain steady state turnover of its epithelium in healthy ocular conditions and
LEC is the potential reservoir of limbal SC. Although a specific limbal SC marker
has yet to be elucidated, our findings have identified several genes of interest,
which will be further studied as candidate genes to validate their potential as stem
cell markers.

Methods

Donor eye tissue preparation

This study was carried out at the Queens Medical Centre, University Hospital Nottingham,
England with approval of Nottingham Research Ethics Committee (REC NO: OY030202).
Protocol was consistent with Tenets of Declaration of Helsinski. Informed, written
consent was obtained from relatives of all the donors. The eyes were harvested within
48 hours of death under aseptic conditions using conventional techniques in order
to maintain the RNA viability. Four pairs of human donor eyes were collected for microarray
study. The inclusion criteria were: i) donors aged between 20 to 70 years; ii) donors
of either sex; iv) Eyes with intact and undamaged OS epithelium, confirmed with dissecting
microscope and patient history as ascertained from the case notes. The corneoscleral
button was dissected from the cadaver eye and processed for sectioning using established
techniques within our department [12]. Briefly, a 15 mm corneal button was trephined maintaining a 3 mm frill of conjunctiva
around the limbus and divided into eight triangular radial segments. Each segment
was positioned in the optimum temperature compound (OCT, Emitech Ltd, East Sussex,
England) with the long dissected edge parallel to the OCT surface and then gradually
frozen using isopentane precooled in liquid Nitrogen. The frozen tissue blocks were
stored at -80°c for future cryosectioning.

Processing of Standard probe samples

Depending on the number of biological samples and their replicates sufficient amount
of control samples are required for any microarray study. As it was not possible to
generate required amount of control sample with LMD, we followed the reference probe
hybrid approach for sample processing as described by Neal and Westwood [86]. Briefly, the reference samples were prepared by pooling the corneal and conjunctival
epithelial RNA. The tissue for RNA extraction was obtained by scrapping the OS epithelial
regions from the cadaver eyes. This approach generated sufficient amount of reference
samples for the microarrays, facilitated better comparison between the two regional
arrays and also highlighted the variations in gene expression between the biological
replicates but not the reference samples. For real time PCR, RNA extraction for each
OS region was performed in triplicate from three different cadaver eyes using RNeasy
Microkit (Qiagen, Crawley, West Sussex, UK) according to manufacturer's protocol.

Laser Microdissection (LMD)

Under RNase and DNase free conditions, 6-7 μm serial sections of frozen tissue blocks
of corneoscleral buttons were prepared with Jung CM 1900 cryostat (Leica, UK) and
examined under light microscope for presence of LEC and also for good epithelial histology
of other OS regions. Prior to LMD, sections were placed on poly-L-lysine coated PALM® membrane slides, fixed in precooled 70% v/v ethanol for 5 minutes and air dried. Following
which, the sections were briefly stained with 0.1% w/v Toludine Blue for 30 seconds,
washed in DEPC treated water and air dried. LMD was performed with the PALM® Microbeam systems (Zeiss Instruments, Bernreid, Germany), using the Robo LPC laser
function, according to manufacturer's recommended guidelines. The area of interest
was cut and catapulted in the caps of collection tubes, coated with special adhesive.
Thereafter RLT RNA lysis buffer (QIAGEN) was added to the collection tubes and stored
at -80°c until further use. For each of the 4 eyes, multiple LMD samples were collected
from five regions creating 5 independent experimental groups; 1) LEC; 2) limbus; 3)
cornea; 4) LEC stroma, and 5) conjunctiva.

Total RNA Extraction

For microarrays total RNA extraction from LMD sections for samples and reference sample
was performed with RNeasy kit, including DNase treatment, according to manufacturer's
protocols (QIAGEN House, West Sussex, UK). RNA quantity and quality was measured with
Picoassay 2100 Bioanalyzer (Agilent Technologies, USA). Samples with concentration
ranging between 20-90 pg/μl and an average RIN value (RNA Integrity Number) of 5.1
[32] were used for further analysis.

Preparation of reference sample for Standard Probe

1 μg of total RNA from each corneal and conjunctival reference sample was mixed and
ethanol precipitated by adding 0.1 volumes Sodium Acetate and 2.5 volumes 100% ETOH
followed by incubation at -20°c for 30 minutes. Samples were then centrifuged at 21000
× g at 4°c for 15 minutes and the pellet washed in 250 μl of 70% v/v ETOH before recentrifugation
at 21000 × g for 5 min. The RNA pellet was dried and resuspended in 10 μl ultra pure,
RNase free water followed by quantitation using NanoDrop ND-1000 UV-Vis Spectrophotometer
(Labtech International Ltd-UK).

RNA Amplification, labelling of the samples and reference sample

Each RNA sample and reference sample was further processed for microarray analysis.
To 0.2 ng/10 μl of starting RNA, 0.5 μl of 1:5000 diluted spike control (GE Healthcare
life Sciences Universal Score card oligonucleotide control), which are sequences from
E. coli genes, was added for validation of microarray data. Complimentary RNA (cRNA) amplification
was performed with Amino Allyl Message Amp™ II aRNA Amplification kit and labelled
with Cy3 and Cy5 reactive dye [Ambion, (Europe) LTD, UK], the Frequency of Incorporation
(FOI) of the Cy3 and Cy5 dye in the labelled samples was measured, according to manufacturer's
protocols. Quality control of the purified cRNA samples was performed at the end of
1st and 2nd rounds by NanoDrop spectrophotometer. Unsatisfactorily amplified and labelled samples
were discarded and new samples were processed.

Microarray hybridisation

500 ng of a labelled sample and reference sample with matching FOI were separately
blocked with 2 μl Poly A and 2 μl human Cot 1 DNA and then combined. This was followed
by ethanol precipitation to generate 2 μl of the hybrid probe in nuclease free water.
Prior to hybridisation, the 30K Human spotted oligonucleotide glass arrays manufactured
in house, (Post-Genomics technologies facility, University of Nottingham, UK), were
blocked with appropriate buffers (5× SSC, 0.2% w/v SDS, 1% w/v BSA), (SSC buffer:
Saline, Sodium Citrate buffer; SDS buffer: Sodium Dodecyl. Sulphate; BSA: Bovine Serum
Albumin). The slides were then washed thrice with ultra pure water, 100% ETOH and
spun dried. For hybridisation, 100 μl of pre warmed Schott 1× Hybridisation Buffer
was gently added to the hybrid probe, heated to 95°C, 2 minutes followed by hybridisation
on automated hybridisation station TECAN HS 4800 (Tecan UK Ltd) using manufacturers
protocols. The conditioned hybridisation station was primed with ultra pure water
followed by a 1 min and a 15 min wash in 5× SSC, 0.2% w/v SDS, at 50°C. Next, 100
μl of probe was injected onto the slide and hybridised with agitation at 50°C for
16 hours, followed by sequential washing in four cycles of 2× SSC, 0.1% SDS, 2 cycles
of 1× SSC, 0.1% SDS, and 0.1× SSC, 0.2% SDS at 40°C, 2 cycles of 0.1× SSC at 23°C,
followed by a final cleaning cycle of ultrapure water before drying. On completion
of the programme, the slides were covered to protect against light and scanned immediately.

Scanning and Data analysis

Hybridised microarray slides were scanned to obtain two coloured digital images on
an Agilent BA scanner. The images were further analysed with Gene Pix Pro 6.0 software.
Poor spots and spots overlying areas of high background intensities were lassoed and
removed from further analysis. However, spots of varying sizes but with good intensities
were included in analysis. The raw data was uploaded on BASE (Bio Array Software Environment; (Lund University, Lund, Sweden) which is a MIAME
(Minimum Information about a Microarray Experiment) compliant system [87]. The raw data sets were expressed as log ratio of channel 1/channel 2 intensities
or log ratio of Cy5/Cy3 (sample/standard probe). Bioassay sets created from the raw
data sets were further filtered to refine the data by removing 'noise'. Following
filtrations, intra-array Lowess normalisation was performed on BASE, and the bioassay sets were then imported to J-Express Pro software (http://www.molmine.comwebcite; MolMine AS, Norway). In J-Express inter array scale normalisation was performed on the imported data sets. Further statistical
analysis of the data was performed in J-Expresspro using a feature subset selection
algorithm (FSS) for two unpaired groups comparison with following parameters: P value
was selected for score method, individual ranking of the genes was performed and fold
change values were log(2) transformed.

Significance Analysis of Microarrays (SAM)

Differentially expressed gene list was generated from the FSS derived gene list (p
value ≤ 0.05) by performing SAM on the data to determine the fold change and the False
Discovery Rate (FDR). Cutoff limit of FDR was set to ≤ 5% and genes with FDR above
this limit were excluded from the analysis. Following SAM the five individual gene
lists generated for each of the OS regions were merged to form an overlapping gene
list for each region.

Principal Component Analysis (PCA)

PCA evaluates variation between the samples. The 2D plots representing the samples
(Figure 2 left) and the genes (Figure 2 right) were generated following FSS analysis in J-Expresspro. The variance of axes
was displayed in percentage. The data with greatest variation is clustered at the
first principal component axis.

Gene Ontology (GO)

The enriched GO terms for each region was determined by uploading the significant
gene list for each region individually on the Database for Annotation, Visualization,
and Integrated Discovery (DAVID) v6.7, 2008, (http://david.abcc.ncifcrf.govwebcite) and the data was analysed according to published methodology [88,89]. GO terms associated with the biological processes (GO~BP) were mostly considered
for analysis. Statistical significance of the GO terms was established by Fishers
Exact T test. GO terms with p values < 0.05 were considered statistically significant.

Canonical pathways on Ingenuity Pathway Analysis

The microarray data was also analysed with Ingenuity Pathways Analysis (IPA) version 7.6 (Ingenuity® Systems, http://www.ingenuity.comwebcite). IPA identified the canonical signalling and metabolic pathways from the IPA library
that were most significant to the data set. The significance of the association between
the data set and the canonical pathway was measured as a ratio and the p value calculated
with Fischer Exact test. The data discussed in this publication have been deposited
in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession
number GSE19035 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19035webcite.

Quantitative Gene Expression Analysis (Real-time PCR)

The relative quantification of mRNA for genes of interest on OS epithelium (LEC, limbus,
cornea, and conjunctiva) was performed with real-time PCR. Approximately 1 ng/μl concentration
of RNA was used for cDNA synthesis using QuantiTect Reverse Transcription Kit (QIAGEN)
according to manufacturer's protocol. Inventoried Taqman assays (Applied Biosystems,
Foster City, CA) were used for selected genes of interest. Each reaction was performed
in triplicate with final reaction volume of 20 μl. The reaction components comprised
of, 10 μl 2× Taqman Gene Expression Master Mix (Applied Biosystems), 1 μl of 20× Taqman
Assay probes (Applied Biosystems), 1 μl cDNA (1:2 dilutions), 8 μl nuclease free water
(Promega UK, Southampton, UK). Non template, reverse transcriptase negative and positive
cDNA from Universal Human Reference RNA (Stratagene, La Jolla, CA) were run as controls.
Amplification was performed on the Mx3005P multicolour 96 well PCR system (Stratagene
Europe, Amsterdam, Netherlands) with the following parameters, 50°C for 2 min and
then 95°C for 10 min followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute.
Data analysis was performed on Mxpro ver 4.2 software to measure the threshold cycle
(Ct) for each reaction. Set of triplicate Ct values for each sample was averaged.
Normalisation of the average sample gene expression was performed with the average
Ct value of 18S rRNA endogenous gene control for that sample.

Statistical Analysis of Real time PCR Data

The real time PCR data was statistically analysed on SPSS ver 16. The average normalised Ct values of gene of interest for each of the OS regions
were subjected to Levene's test, to measure the equality of variance. The normally
distributed data was then analysed with one way analysis of variance (ANOVA) with
Bonferonni's posthoc test. Non parametric distributed samples were analysed by Kruskal-Wallis
test followed by Mann-Whitney test, p value < 0.05 was considered statistically significant.

Authors' contributions

BBK was involved in sample collection and processing and also performed microarray,
real-time PCR and immunohistochemistry experiments and its analysis. HSD and PJT helped
and supervised over the analysis and upload of microarray data on GEO profile. HSD
conceived the study and HSD, PJT, BBK, DGP, AH and VAS participated in its design.
HSD and AH had supervisory role and looked after the administration and financial
aspect of the project. BBK created a draft of the manuscript, HSD, PJT, AH, and IM
helped in editing the manuscript. DGP is supervisor for lasermicrodissection and advised
on performing the procedure. IM had advised on data analysis and trouble shooting
of real time PCR experiments. AMY helped with immunofluorescence experiment. All authors
have read and approved the manuscript.

Acknowledgements

I gratefully acknowledge financial support to this project from Royal College of Surgeons
of Edinburgh, UK. I would like to thanks Toshana Foster and Stacy Mutch (research
technicians) for their support with microarray experiments. I wish to thanks Mr K
Tsintzas, School of Biomedical sciences, University of Nottingham, UK, for his helpful
advice on real-time PCR data analysis.